Acute vascular diseases, such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other blood system thromboses constitute major health risks. Such diseases are caused by either partial or total occlusion of a blood vessel by a blood clot, which contains fibrin and platelets.
Thrombin is the naturally occurring protein which catalyzes the conversion of fibrinogen to fibrin, the final step in blood clot formation. In addition to catalyzing the formation of a fibrin clot, thrombin also activates platelet aggregation and release reactions. This means that thrombin plays a central role in both acute platelet-dependent (arterial) thrombosis [S. R. Hanson and L. A. Harker, "Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic Antithrombin D-Phenylalanyl-L-Prolyl-L-Arginylchloromethylketone", Proc. Natl. Acad. Sci. USA, 85, pp. 3184-88 (1988)] and fibrin-dependent (venous) thrombosis.
Thrombin has several other bioregulatory roles [J. W. Fenton, II, "Thrombin Bioregulatory Functions", Adv. Clin. Enzymol., 6, pp. 186-93 (1988)]. For example, thrombin can directly activate an inflammatory response by stimulating the synthesis of platelet activating factor (PAF) in endothelial cells [S. Prescott et al., "Human Endothelial Cells in Culture Produce Platelet-Activating Factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) When Stimulated With Thrombin", Proc. Natl. Acad. Sci. USA, 81, pp. 3534-38 (1984)]. PAF is exposed on the surface of endothelial cells and serves as a ligand for neutrophil adhesion and subsequent degranulation [G. M. Vercolletti et al., "Platelet-Activating Factor Primes Neutrophil Responses to Agonists: Role in Promoting Neutrophil-Mediated Endothelial Damage", Blood, 71, pp. 1100-07 (1988)]. Alternatively, thrombin may promote inflammation by increasing vascular permeability which can lead to edema [P. J. Del Vecchio et al., "Endothelial Monolayer Permeability To Macromolecules", Fed. Proc., 46, pp. 2511-15 (1987)]. Reagents which block the active site of thrombin, such as hirudin, interrupt the activation of platelets and endothelial cells [C. L. Knupp, "Effect of Thrombin Inhibitors on Thrombin-Induced Release and Aggregation", Thrombosis Res., 49, pp. 23-36 (1988)].
Thrombin has also been implicated in promoting cancer, based on the ability of its native digestion product, fibrin, to serve as a substrate for tumor growth [A. Falanga et al., "Isolation and Characterization of Cancer Procoagulant: A Cysteine Proteinase from Malignant Tissue", Biochemistry, 24, pp. 5558-67 (1985); S. G. Gordon et al., "Cysteine Proteinase Procoagulant From Amnion-Chorion", Blood, 66, pp. 1261-65 (1985); and A. Falanga et al., "A New Procoagulant In Acute Leukemia", Blood, 71, pp. 870-75 (1988)]. And thrombin has been implicated in neurodegenerative diseases based on its ability to cause neurite retraction [D. Gurwitz et al., "Thrombin Modulates and Reverses Neuroblastoma Neurite Outgrowth", Proc. Natl. Acad. Sci. USA, 85, pp 3440 -44 (1988)]. Therefore, the ability to regulate the in vivo activity of thrombin has many important clinical implications.
One route to the successful treatment or prevention of acute vascular disease is the inhibition of thrombin. Many types of thrombin inhibitors are already known in the art. Heparin, an indirect inhibitor of thrombin, is widely used to treat venous thrombosis. Although effective against fibrin-dependent clot formation, heparin has little efficacy in inhibiting thrombin-induced activation of platelets. Therefore, this drug is not utilized in the treatment of arterial thromboses. Moreover, heparin produces many undesirable side effects, including hemorrhaging and thrombocytopenia.
Hirudin is a naturally occurring polypeptide which is produced by the blood sucking leech Hirudo medicinalis. This compound, which is synthesized in the salivary gland of the leech, is the most potent natural inhibitor of coagulation known. Hirudin prevents blood from coagulating by binding tightly to thrombin (K.sub.d =2.times.10.sup.-11 M) in a 1:1 stoichiometric complex [S. R. Stone and J. Hofsteenge, "Kinetics of the Inhibition of Thrombin by Hirudin", Biochemistry, 25, pp. 4622-28 (1986)]. This, in turn, inhibits thrombin from catalyzing the conversion of fibrinogen to fibrin (clot), as well as inhibiting all other thrombin-mediated processes [J. W. Fenton, II, "Regulation of Thrombin Generation and Functions", Semin. Thromb. Hemost., 14, pp. 234-40 (1988)].
Hirudin inhibits thrombin by binding to the latter at two separate sites. Initially, the C-terminus of hirudin interacts with an "anion-binding exosite" (ABE) in thrombin [J. W. Fenton, II et al., "Thrombin Anion Binding Exosite Interactions with Heparin and Various Polyanions", Ann. New York Acad. Sci., 556, pp. 158-65 (1989)]. Following this low affinity binding, the hirudin-thrombin complex undergoes a conformational change and amino terminal portion of hirudin is able to bind to the catalytic site of thrombin [S. Kono et al., "Analysis of Secondary Structure of Hirudin and the Conformational Change Upon Interaction with Thrombin", Arch. Biochem. Biophys., 267, pp. 158-66 (1988)].
The isolation, purification and amino acid sequence of hirudin are known in the art [P. Walsmann and F. Markwardt, "Biochemical and Pharmacological Aspects of the Thrombin Inhibitor Hirudin", Pharmazie, 36, pp. 653-60 (1981); J. Dodt et al., "The Complete Covalent Structure of Hirudin: Localization of the Disulfide Bonds", Biol. Chem. Hoppe-Seyler, 366, pp. 379-85 (1985); S. J. T. Mao et al., "Rapid Purification and Revised Amino Terminal Sequence of Hirudin: A Specific Thrombin Inhibitor of the Blood-Sucking Leech", Anal. Biochem, 161 pp 514-18 (1987); and R. P. Harvey et al., "Cloning and Expression of a cDNA Coding for the Anti-Coagulant Hirudin from the Bloodsucking Leech, Hirudo medicinalis", Proc. Natl. Acad. Sci. USA, 83, pp. 1084-88 (1986)].
In animal studies, hirudin, purified from leeches, has demonstrated efficacy in preventing venous thrombosis, vascular shunt occlusion and thrombin-induced disseminated intravascular coagulation. In addition, hirudin exhibits low toxicity and a very short clearance time from circulation [F. Markwardt et al., "Pharmacological Studies on the Antithrombotic Action of Hirudin in Experimental Animals", Thromb. Haemost., 47, pp. 226-29 (1982)].
Hirudin has more recently been cloned and expressed in E. coli [European patent applications 158,564, 168,342 and 171,024] and yeast [European patent application 200,655]. Despite these advances, hirudin is still moderately expensive to produce and it is not widely available commercially.
Recently, efforts have been made to identify peptide fragments of native hirudin or derivatives thereof which are also effective in prolonging clotting times. Such compounds are described in European patent application Nos. 276,014,291,982, 333,356, 341,607 and 372,670. The molecules described in these patent applications demonstrated varying efficacy in inhibiting clot formation, but were all 2 to 4 orders of magnitude less potent than hirudin. Such peptide fragments, therefore, may not be fully satisfactory to dissolve blood clots in on-going therapy regimens.
More recently, compounds which mimic the action of hirudin by binding to both the anion binding exosite and the catalytic site of thrombin have been described [U.S. patent application Ser. Nos. 395,482, now abandoned, and 549,388, now U.S. Pat. No. 5,196,404]. These compounds demonstrate thrombin inhibitory activity equal to or greater than native hirudin. They are also smaller than hirudin and therefore less antigenic. These inhibitors are also produced synthetically, allowing for the production of commercially feasible quantities at reasonable costs.
Despite the developments to date, there is an ongoing need for even more potent thrombin inhibitors which can be produced inexpensively and in commercially feasible quantities. Such inhibitors would not only be effective in treating and preventing vascular disease, but may also be therapeutically useful in treating cancer, neurodegenerative disease and inflammation.